1. Field of the Invention
This invention relates generally to the field of vaccine strains. More particularly, it relates to attenuated strains of Francisella tularensis and methods of use thereof.
2. Description of the Related Art
Francisella tularensis (F. tularensis) is a Gram-negative coccobacillus with a natural reservoir that includes small mammals such as rabbits, hares, and rodents, as well as aquatic environments and soil. F. tularensis includes four subspecies—tularensis (type A), holarctica (type B), novicida, and mediasiatica—and the most virulent of these, F. tularensis subspecies tularensis, can cause infection with doses as low as 10 colony forming units (cfu). Transmission typically occurs via handling of infected animals and carcasses, consumption of contaminated food products, and occasionally through insect vectors. The strain and route of infection determines the progression of the disease, which generally involves spread to multiple organ systems and the lymphatic system. The U.S. Government currently classifies F. tularensis as a Tier 1 Select Agent, meaning that it has been determined to potentially pose a severe threat to human and animal health. Interest in the pathobiology of the bacterium has been rekindled with the recognition that F. tularensis may be deployed as a potent bioweapon due to its ease of dissemination via aerosolization and extremely low infective dose. Left untreated, F. tularensis has the potential to be lethal in 30-60% of infected individuals.
F. tularensis enters host macrophages and employs a variety of methods to evade host defense mechanisms throughout the infection cycle. The host immune system is evaded by a lipopolysaccharide (LPS) that interacts poorly with host pattern recognition receptors and does not induce inflammatory cytokines. In serum, F. tularensis binds Factor H to inactivate C3b by converting it to iC3b, which interferes with the formation of a membrane attack complex and opsonizes the bacterium to facilitate pathogen entry into the host by exploiting macrophages as a niche for replication. F. tularensis-containing phagosomes are arrested in the late endosomal stage and avoid fusion to lysosomes. The phagosome is transiently acidified leading to its disruption and escape of the bacteria into the cytosol. After replication in the cytoplasm, the bacteria induce autophagy and are released through apoptosis and pyroptosis.
Despite a generalized understanding of the F. tularensis infective cycle, more insight into the genes contributing to and controlling, for example, infection, intracellular survival, replication, pathogenicity, and effect on host immune response is needed for the development of effective vaccine and therapeutic countermeasures. An undefined vaccine strain of F. tularensis referred to as Live Vaccine Strain (LVS) was developed, which has demonstrated the ability confer at least partial immunity to challenges by F. tularensis subsp. tularensis. However, due to an incomplete understanding of the attenuation mechanism, presence of side effects, and other safety concerns, LVS has not been approved by the U.S. Food and Drug Administration for use as a vaccine.
In addition, a variety of methods such as random insertional mutagenesis using transposons, targeted gene replacement based on homologous recombination strategies, and targeted insertional inactivation using group II introns or targetrons have been used to mutagenize or disrupt F. tularensis genes and evaluate impact on intramacrophage survival and growth. Generation of transposon mutant libraries provide a high-throughput technique to screen for mutations that affect, for example, intramacrophage growth and virulence. However, the random nature of transposon insertions requires additional steps to ensure lack of hot spots and even distribution of insertions, as well as to determine the site of insertion and to demonstrate that a single insertion is responsible for the observed effects. Homologous recombination provides precise, targeted inactivation of genes but is difficult to adapt to high-throughput format. Use of targetrons combines the best aspects of transposon and homologous recombination techniques to provide a highly precise and easily adaptable method for disrupting and/or inactivating large numbers of genes throughout the genome.